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Structural analysis of pathogenic mutations targeting Glu427 of ALDH7A1, the hot spot residue of pyridoxine-dependent epilepsy.

ABSTRACT: Certain loss-of-function mutations in the gene encoding the lysine catabolic enzyme aldehyde dehydrogenase 7A1 (ALDH7A1) cause pyridoxine-dependent epilepsy (PDE). Missense mutations of Glu427, especially Glu427Gln, account for ~30% of the mutated alleles in PDE patients, and thus Glu427 has been referred to as a mutation hot spot of PDE. Glu427 is invariant in the ALDH superfamily and forms ionic hydrogen bonds with the nicotinamide ribose of the NAD⁺ cofactor. Here we report the first crystal structures of ALDH7A1 containing pathogenic mutations targeting Glu427. The mutant enzymes E427Q, Glu427Asp, and Glu427Gly were expressed in Escherichia coli and purified. The recombinant enzymes displayed negligible catalytic activity compared to the wild-type enzyme. The crystal structures of the mutant enzymes complexed with NAD⁺ were determined to understand how the mutations impact NAD⁺ binding. In the E427Q and E427G structures, the nicotinamide mononucleotide is highly flexible and lacks a defined binding pose. In E427D, the bound NAD⁺ adopts a "retracted" conformation in which the nicotinamide ring is too far from the catalytic Cys residue for hydride transfer. Thus, the structures revealed a shared mechanism for loss of function: none of the variants are able to stabilise the nicotinamide of NAD⁺ in the pose required for catalysis. We also show that these mutations reduce the amount of active tetrameric ALDH7A1 at the concentration of NAD⁺ tested. Altogether, our results provide the three-dimensional molecular structural basis of the most common pathogenic variants of PDE and implicate strong (ionic) hydrogen bonds in the aetiology of a human disease.

SUBMITTER: Laciak AR

PROVIDER: S-EPMC7182499 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Pyridoxine-dependent epilepsy was recently shown to be due to mutations in the ALDH7A1 gene, which encodes antiquitin, an enzyme that catalyses the nicotinamide adenine dinucleotide-dependent dehydrogenation of l-alpha-aminoadipic semialdehyde/L-Delta1-piperideine 6-carboxylate. However, whilst this is a highly treatable disorder, there is general uncertainty about when to consider this diagnosis and how to test for it. This study aimed to evaluate the use of measurement of urine L-alpha-aminoadipic semialdehyde/creatinine ratio and mutation analysis of ALDH7A1 (antiquitin) in investigation of patients with suspected or clinically proven pyridoxine-dependent epilepsy and to characterize further the phenotypic spectrum of antiquitin deficiency. Urinary L-alpha-aminoadipic semialdehyde concentration was determined by liquid chromatography tandem mass spectrometry. When this was above the normal range, DNA sequencing of the ALDH7A1 gene was performed. Clinicians were asked to complete questionnaires on clinical, biochemical, magnetic resonance imaging and electroencephalography features of patients. The clinical spectrum of antiquitin deficiency extended from ventriculomegaly detected on foetal ultrasound, through abnormal foetal movements and a multisystem neonatal disorder, to the onset of seizures and autistic features after the first year of life. Our relatively large series suggested that clinical diagnosis of pyridoxine dependent epilepsy can be challenging because: (i) there may be some response to antiepileptic drugs; (ii) in infants with multisystem pathology, the response to pyridoxine may not be instant and obvious; and (iii) structural brain abnormalities may co-exist and be considered sufficient cause of epilepsy, whereas the fits may be a consequence of antiquitin deficiency and are then responsive to pyridoxine. These findings support the use of biochemical and DNA tests for antiquitin deficiency and a clinical trial of pyridoxine in infants and children with epilepsy across a broad range of clinical scenarios.

Project description:BackgroundLittle is known about pyridoxine-dependent epilepsy due to α-aminoadipic semialdehyde dehydrogenase deficiency (PDE-ALDH7A1) in adulthood, as the genetic basis of the disorder has only been elucidated 15 years ago. This creates a knowledge gap for physicians, pediatric patients and their parents, which was aimed to address in this study using clinical data as well as patient-reported outcome measures (PROMs) for the patient's perspective.MethodsDutch, genetically confirmed PDE-ALDH7A1 patients ≥18 years were eligible for inclusion. Clinical data were collected as well as PROMs (PROMIS item banks Anxiety, Depression, Anger, Physical Functioning, Cognitive Functioning, Cognitive Abilities, Ability to Participate and Satisfaction with Social Roles).ResultsTen out of 11 patients agreed to participate (91% response rate). Seizure control at last follow up (median age 25.2 years, range 17.8-29.8 years) was achieved with pyridoxine monotherapy in 70%, 20% with adjunct common-anti epileptic drugs and 10% did not obtain complete seizure control. Neurologic symptoms were present in all but one patient (90%) and included tremors, noted in 40%. Neuro-imaging abnormalities were present in 80%. Intellectual disability was present in 70%. One patient (10%) attended university, three maintained a job without assistance, five maintained a job with assistance or attended social daycare, and one patient never followed regular education. The cohort scored significantly lower on the PROMIS Cognitive Functioning compared to the general (age-related) population. Distribution of scores was wide on all PROMIS item banks.Discussion & conclusionOutcomes of this young adult cohort are heterogeneous and individualized approaches are therefore needed. Long-term seizure control with pyridoxine was achieved for almost all patients. Neurologic symptoms were noted in the majority, including tremors, as well as neuro-imaging abnormalities and intellectual disability, additionally reflected by the PROMIS Cognitive Functioning. PDE-ALDH7A1 patients scored comparable to the general population on all other PROMs, especially regarding Ability to Participate and Satisfaction with Social Roles this may indicate a positive interpretation of their functioning. The aim is to expand this pilot study to larger populations to obtain more solid data, and to advance the use of PROMs to engage patients in research and provide the opportunity for personalized care.

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Structural analysis of pathogenic mutations targeting Glu427 of ALDH7A1, the hot spot residue of pyridoxine-dependent epilepsy.

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